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Mouse tumor models have been successfully used to generate preclinical data for numerous clinical programs including immunotherapy. Preclinical in vivo studies are typically carried out using young mice (often less than 2 months old) to generate efficacy data, predictive biomarker and pharmacodynamic markers. In notable contrast, the majority of human cancer occur in adult and older patients. It has become increasingly clear that the immune system of young and old mice is quite different with regards to the relative abundance and functionality of different cell populations. Data generated using young mice could provide a distorted assessment of the potential activity of immuno-oncology drugs in the clinic. Therefore, we tested the activity of GITRL-Fc protein in both young and old mice (>9 months). Previously using extensive young mice experiments, we have shown that GITRL-Fc promoted a robust antitumor immune response and enhanced tumor-specific T-cell responses, particularly of the Th1 type, and also led to a reduction in Treg-mediated immunosuppressive activity. Compared to young mice, tumors grew faster in older mice, and peripheral blood of older tumor-bearing mice has fewer T cells and NK cells. The total MDSC population was increased in the blood and spleen of old tumor-bearing mice, with a significantly higher number of G-MDSCs in the blood. On the other hand, old mice had reduced “antigen-presenting cells” (macrophages/dendritic cells expressing MHCII) in the blood. Furthermore, splenocytes from old mice had impaired production of IL-2. GITRL-Fc significantly inhibited tumor growth in both older and younger mice. However, efficacy was more pronounced in young mice, which frequently exhibited complete tumor regression. There were fewer tumor-infiltrating immune cells with less CD8 T and NK cells in older mice compared to young mice, consistent with faster tumor growth. Interestingly, GITR expression in CD8 T cells in old mice was lower compared with young mice at the tumor site. In old mice, GITRL-Fc (mIgG2a) was still able to deplete Tregs in tumor and increase Tregs in the spleen as has been previously shown with GITRL-Fc in young mice. On the other hand, GITRL-Fc deficient in effector function (mIgG2a (N297A)) did not deplete Tregs in the tumor but did retain some antitumor growth activity, indicating a role for GITR signaling in the mechanism of efficacy by GITLR-Fc. In conclusion, the results demonstrate the potential for the aging of the immune system to impact the efficacy observed with immunotherapy agents and highlight the potential benefits of conducting efficacy studies with both young and older mice. Citation Format: Angie Inkyung Park, Minu K. Srivastava, Rui Yun, Jenny Pokorny, Janice Yu, Fumiko Axelrod, Austin Gurney. Effect of aging on the antitumor activity of GITRL-Fc [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 70.

T cell immunoreceptor with Ig and ITIM domains (TIGIT) is a co-inhibitory molecule containing an immunoreceptor tyrosine-based inhibition motif (ITIM) within its cytoplasmic tail, and is highly expressed on regulatory T cells and activated CD4+ T, CD8+ T, and NK cells. TIGIT competes with CD226, which contains an immunoreceptor tyrosine-based activation motif (ITAM) within its cytoplasmic tail for ligands poliovirus receptor (PVR) and poliovirus receptor-related 2 (PVRL2), with higher affinity to PVR. The ligands are expressed on the surface of antigen presenting cells and at high levels on most tumors. Therefore, when TIGIT is present, the ligands preferentially engage TIGIT rather than CD226, leading to cell suppression. We have generated antibodies against TIGIT that blocks ligand binding and inhibits TIGIT signaling. The clinical candidate, OMP-313M32 binds human TIGIT but not rodent and non-human primate TIGIT. Therefore, a surrogate antibody was generated for pre-clinical assessments in mice. Antibody 313R12 is an anti-mouse TIGIT antibody that can block mouse PVR ligand binding and inhibit TIGIT signaling in a manner similar to the clinical candidate OMP-313M32. 313R12 inhibited the growth of syngeneic colon and kidney tumors in immune competent mice. In some cases, anti-TIGIT antibody 313R12 caused complete tumor regression and a potent anti-tumor immune memory response as demonstrated by the lack of tumor growth upon re-challenge of mice that remained tumor-free after prior anti-TIGIT treatment. Mechanistically, anti-TIGIT antibody 313R12 was shown to induce a Th1 response and increase cytotoxic T lymphocyte (CTL) activity. By in vivo depletion of T cell populations, we have shown that CD8 T cell depletion completely abrogated the anti-TIGIT therapeutic effect, whereas CD4 T cell depletion led to partial reversal of efficacy of anti-TIGIT. Therefore, both CD4+ and CD8+ T cells are critical for anti-TIGIT-mediated immune responses. Using mice reconstituted with human hematopoietic stem cells, we also demonstrated that the clinical candidate OMP-313M32 inhibits patient-derived melanoma tumor growth. Taken together, these data demonstrate that anti-TIGIT therapy suppresses tumor growth and generates long-term immunological memory against multiple tumors. Citation Format: Angie Inkyung Park, Minu Srivastava, Erin Mayes, Hyun-Bae Jie, Rui Yun, Christopher Murriel, Ming-hong Xie, Andrew Lam, May Ji, Fumiko Axelrod, Jorge Monteon, John Lewicki, Tim Hoey, Austin Gurney. Antibody against TIGIT (T cell immunoreceptor with Ig and ITIM domains) induces anti-tumor immune response and generates long-term immune memory [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2003. doi:10.1158/1538-7445.AM2017-2003

TIGIT (T cell immunoreceptor with Ig and ITIM domains) has been recently described as an inhibitory receptor which blocks CD8 T cell-mediated anti-tumor immune responses. We have generated an anti-mouse TIGIT antibody (313R12) to evaluate drug efficacy and mechanism of action in pre-clinical tumor models. Anti-TIGIT as a single agent promoted an anti-tumor immune response in multiple syngeneic mouse tumor models. Anti-TIGIT enhanced tumor specific T cell responses, particularly of the Th1 type and reduced Th2 type responses and also increased the function of cytotoxic T cells. Furthermore, anti-TIGIT displayed combination activity with immune checkpoint inhibitors anti-PD1 and anti-PDL1 in inhibiting tumor growth, promoting complete tumor rejection and significantly increasing mouse survival in the murine CT26 colon carcinoma model as compared to controls and single agents alone. Mice “cured” with anti-TIGIT/anti-PDL1 or anti-TIGIT/anti-PD1 combination treatments did not form tumors upon subsequent re-challenges with increasing number of CT26 tumor cells, suggesting the existence of immunologic memory. IL2 and tumor-specific IFN-γ production by splenic T cells were increased in mice who responded to combination treatment compared to controls. Additionally, both effector and memory CD8+ T cell frequencies were increased within the total CD8+ T cell population in responding mice. We also demonstrated a systemic increase in tumor-specific CD8 T cells after anti-TIGIT/anti-PDL1 combination treatment compared to controls. Therefore, these results suggest that co-targeting of TIGIT and PD1 or PDL1 may be an effective and durable cancer therapy by increasing T cell-mediated anti-tumor immune responses and promoting long-term immunological memory. Citation Format: Minu K. Srivastava, Rui Yun, Erin Mayes, Janice Yu, Hyun-Bae Jie, Fumiko Axelrod, Ming-Hong Xie, Jorge Monteon, Andrew Lam, May Ji, Yuwang Liu, John Lewicki, Tim Hoey, Austin Gurney, Angie Inkyung Park. Anti-Tigit induces T cell mediated anti-tumor immune response and combines with immune checkpoint inhibitors to enhance strong and long term anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2612. doi:10.1158/1538-7445.AM2017-2612

Although the interaction between GITR (Glucocorticoid-Induced Tumor Necrosis Factor Receptor) and GITRL (its ligand) is important for the development of immune responses, the cellular mechanisms underlying anti-tumor immunity including both innate and adaptive immunity is not fully understood. We generated a novel single-gene linkerless GITRL trimer fused to an immunoglobulin Fc domain (GITRL-Fc), and investigated its effect on controlling tumor growth and immune responses in preclinical tumor models. Treatment with GITRL-Fc significantly reduced tumor growth in several preclinical tumor models by inducing Th1 biased anti-tumor immunity and reducing Treg-mediated immune suppression. Immune cell depletion studies showed that anti-tumor immunity induced by GITRL-Fc depended on CD8+ T cells as well as NK cells. Furthermore, the combination of GITRL-Fc with PD-1 blockade significantly reduced the tumor growth in the Renca murine kidney adenocarcinoma tumor model. Taken together, these results suggest that GITRL-Fc can improve cancer treatment as a single agent or in combination therapy by enhancing innate and adaptive cellular immunity. Citation Format: Hyun-Bae Jie, Minu K. Srivastava, Erin Mayes, Rui Yun, Fumiko Axelrod, Jorge Monteon, Austin Gurney, Angie In-Kyung Park. GITRL-Fc can significantly reduce tumor growth by stimulating innate and adaptive immunity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3215.

Blocking DLL4, a Notch ligand, effectively inhibits tumor growth by increasing non-functional angiogenesis and decreasing the cancer stem cells (CSC) population. We are currently testing an anti-DLL4 antibody, demcizumab, in Phase1B trials in NSCLC, pancreatic, and ovarian cancer. DLL4 is also known to modulate immune responses. In the current study we examine the impact of anti-DLL4 on anti-tumor immune responses as a single agent and in combination with the key immune checkpoint inhibitor Programmed Cell Death Protein 1 (PD1). While the recent clinical success of PD1 inhibitors represents a new and promising cancer immunotherapeutic approach, high initial response rates are often associated by a lack of long-term, durable effects in a significant number of patients. Therefore, we hypothesized that dual blockade of DLL4 and PD1 might further impact tumor growth by further enhancing anti-tumor immune immunity. Our data demonstrates that dual blockade of DLL4 and PD1 using antibodies not only reduces tumor growth, but also led to tumor rejection in ∼50% in CT26WT tumor-bearing mice, similar to those treated with anti-PD1 alone (no tumor rejection was observed with anti-DLL4 alone). Anti-PD1 increased specific CD8+ T cell-mediated IFN-γ production while decreasing IL6. Anti-DLL4 treatment reduced IL17 production. Interestingly, only the dual blockage led to increased production of IL2 by splenocytes. Since IL2 is required for secondary population expansion of CD8+ memory T cells, increased IL2 in the combination group suggests potential for increased T cell activation, maintenance and memory T cell function, as compared to single agent anti-DLL4 and anti-PD1. While anti-PD1 reduced inhibition of CD4+ T cell proliferation by Tregs, the dual blockade significantly reduced Treg-mediated CD8+ T cell suppression. Furthermore, both effector and memory CD8+ T cell frequencies were increased within the total CD8+ T cell population. Interestingly, anti-PD1 decreased granulocytic MDSCs, while anti-DLL4 reduced monocytic MDSCs. Mice cured with single-agent anti-PD1 and anti-DLL4/anti-PD1 combination treatments were protected from series of re-challenge with tumor cells, suggesting the existence of immunologic memory. Interestingly, more mice were protected from tumor re-challenge when both DLL4 and PD1 were blocked, as compared to PD1 alone. Surprisingly, mice previously treated with the anti-DLL4/anti-PD1 combination produced more IL2, clearly indicating the role of DLL4 blockade in enhancing anti-tumor immunity. Therefore, these results show that dual targeting of DLL4 and PD1 may be an effective and durable cancer therapy by increasing anti-tumor immune response and promoting long-term immunological memory. Citation Format: Minu Srivastava, Christopher L. Murriel, Julie Roda, Hyun-Bae Jie, Fumiko Axelrod, Ming-Hong Xie, Rui Yun, Erin Mayes, Trevor Bentley, Belinda Cancilla, Raymond Tam, Tracy Tang, Ann Kapoun, John Lewicki, Tim Hoey, Austin Gurney, Angie Inkyung Park. Dual targeting of Delta-like ligand 4 (DLL4) and programmed death 1(PD1) inhibits tumor growth and generates enhanced long-term immunological memory. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 255. doi:10.1158/1538-7445.AM2015-255